PPARγ; COX-2; NF-κB

Adelmidrol is a derivative of palmitoylethanolamide. Adelmidrol lowers the levels of nitrotyrosine and poly(ADP)ribose in the colon, as well as NF-κB translocation, COX-2, and p-ERK expression.

Adelmidrol (10 mg/kg, o.s.) significantly lessens both the severity and extent of the macroscopic and histologic signs of colon injury. Additionally, all mice experience diarrhea and lose weight (in comparison to the sham groups) 4 days after being treated with dinitrobenzene sulfonic acid (DNBS), which causes colitis. Treatment with adelmidrol (10 mg/kg, o.s.) greatly slows down the loss of body weight. The inflammatory bowel disease (IBD) brought on by DNBS intrarectally administered is also characterized by an increase in myeloperoxidase (MPO) activity, a sign of neutrophil accumulation in the colon. This is consistent with light microscopic findings that a significant amount of neutrophils were present in the colon of vehicle-treated DNBS mice. Adelmidrol (10 mg/kg, o.s.) on the other hand significantly lowers the level of polymorphonuclear cell infiltration in the inflamed colon (measured as a decrease in MPO activity)[1].

The levels of Ikb-α, nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), phosphoextracellular signal-regulated kinase (p-ERK), Bcl-2, Bax, lamin a/c, and β-actin were calculated, as previously described, in cytosolic and nuclear fractions from colon tissue collected at the end of the experiment with minor modifications. Colon tissue from each mouse was suspended in extraction buffer A containing 0.2 mM phenylmethylsulfonyl fluoride, 0.15 mM pepstatin A, 20 mM leupeptin, 1 mM sodium orthovanadate, homogenized at the maximum setting for 2 minutes, and centrifuged at 12,000g × rpm for 4 minuteS at 4°C. Supernatants represented the cytosolic fraction. The pellets, containing enriched nuclei, were resuspended in buffer B containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 20 mM leupeptin, 0.2 mM sodium orthovanadate. After centrifugation 10 minutes at 12,000 rpm at 4°C, the supernatants containing the nuclear protein were stored at −80°C for further analysis. The filters were blocked with 1× PBS, 5% (w/v) nonfat dried milk for 40 minutes at room temperature, and successively probed with anti-Ikb-α (1/500 in PBS, v/v, Santa Cruz Biotechnology), anti-NF-κB (1/500 in PBS, v/v, Santa Cruz Biotechnology), anti-COX-2 (1/500 in PBS, v/v, Cayman), anti-p-ERK (1/500 in PBS, v/v, Santa Cruz Biotechnology), anti-Bax (SantaCruz Biotechnology 1/500 in PBS, v/v), anti-Bcl-2 (1/500 in PBS, v/v, Santa Cruz Biotechnology), and anti-lamin a/c (1/500 in PBS, v/v, Santa Cruz Biotechnology) in 1× PBS, 5% (w/v) nonfat dried milk, 0.1% Tween-20 (PMT) at 4°C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research, West Grove, PA) for 1 hour at room temperature. To establish that blots were loaded with equivalent amounts of protein lysates, they were similarly incubated with antibody against β-actin (1/1000 in PBS, v/, Santa Cruz Biotechnology v). Relative expression of the protein bands for Ikb-α (37 kDa), NF-κB (65 kDa), COX-2 (72 kDa), p-ERK (46 kDa), Bcl-2 (29 kDa), Bax (23 kDa), lamin a/c (65 kDa), and β-actin (42 kDa) were detected with the enhanced chemiluminescence detection system according to the manufacturer’s instructions (SuperSignal West Pico Chemiluminescent Substrate, Thermo Fisher Scientific, Waltham, MA). Expression of protein bands was calculated by densitometry with Bio-Rad ChemiDoc XRS + software (Hercules, CA) and standardized to β-actin levels. Images of blot signals (8-bit/600-dpi resolution) were imported to an analysis program (Image Quant TL, v2003). Commercially available molecular weight markers (10–250 kDa) were used to establish molecular weight positions.

According to a previously described technique, tissue myeloperoxidase (MPO) activity was measured using a spectrophotometric assay with tetramethylbenzidine as the substrate to examine neutrophil infiltration in the colon. Colon tissues were gathered and weighed following DNBS injection. Each piece of tissue was homogenized in a solution containing 0.5% hexadecyltrimethyl ammonium bromide dissolved in 10 mM potassium phosphate buffer, pH 7, and centrifuged for 30 minutes at 20,000g at 4°C. The supernatant was then allowed to react with a solution containing 1.6 mM tetramethylbenzidine and 0.1 mM H2O2. At 650 nm, the magnitude of the absorbance change was measured spectrophotometrically. MPO activity was defined as the amount of enzyme able to break down 1 mmol of peroxide per minute at 37 °C and expressed in U/g wet tissue.

Mice: Male CD1 adult mice weighing 25 to 30 g and male mice weighing 20 to 27 g are kept in a controlled environment on a 12-hour light/dark cycle with unlimited access to food and water. The following groups of mice are randomly assigned (10 per group): (1)Sham+vehicle group: Saline (the vehicle solution) is administered orally for 4 days. (2) O.s. administration of Sham+Adelmidrol (10 mg/kg) for 4 days. (3) DNBS+vehicle: Mice are given an injection of DNBS as described, and then a vehicle (saline) is administered intravenously every day for four days, beginning 60 minutes after the DNBS injection. (4) DNBS+Adelmidrol (10 mg/kg): Mice are given DNBS injections as directed, and Adelmidrol (10 mg/kg) is administered orally daily beginning 60 minutes after DNBS injections[1].

[1]. Adelmidrol, a Palmitoylethanolamide Analogue, as a New Pharmacological Treatment for the Management of Inflammatory Bowel Disease. Mol Pharmacol. 2016 Nov;90(5):549-561.

Adelmidrol is under investigation in clinical trial NCT01420510 (Saginil in vs. Placebo for Gynecologic Oncology Patients Affected by Vaginitis).

C13H26N2O4

274.36

274.189

C, 56.91; H, 9.55; N, 10.21; O, 23.33

1675-66-7

176874

White to off-white solid powder

1.097g/cm3

596.4ºC at 760mmHg

132-134 ºC

314.5ºC

1.04E-16mmHg at 25°C

1.492

0.715

4

4

12

19

223

0

O=C(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(N([H])C([H])([H])C([H])([H])O[H])=O)N([H])C([H])([H])C([H])([H])O[H]

PAHZPHDAJQIETD-UHFFFAOYSA-N

InChI=1S/C13H26N2O4/c16-10-8-14-12(18)6-4-2-1-3-5-7-13(19)15-9-11-17/h16-17H,1-11H2,(H,14,18)(H,15,19)

N,N'-bis(2-hydroxyethyl)nonanediamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ≥ 30 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.58 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.58 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)